Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.
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Acknowledgements The authors would like to thank to Mrs. Mer-cedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Economía y Competi-tividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/ 00034), Institució Catalana de Recerca i Estudis Avanc¸ats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the ‘‘Neuropharmacology and Pain’’ accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences.
- Active zone
- Calcium channels
- Electron microscopy
- Freeze-fracture replica immunolabelling
- GABA receptors
- Parallel fibre
- Potassium channels
- Purkinje cells